The invention relates to transgenic non-human animals and transgenic non-human animal cells harboring a transgene containing a mutation in the A-myb gene and having a functionally disrupted A-myb gene locus. The invention further relates to transgenes and targeting constructs used to produce such transgenic animals and cells, methods of using such animals for modeling male infertility disorders, and methods for using such animals to produce transgenic nonhuman animals and cells including a further transgene.
The myb gene family currently consists of three members, named A, B and c-myb. Of these, c-myb is the most extensively studied member. The B-myb and A-myb genes share extensive sequence homology with c-myb.
The myb oncogene was first identified as the transforming gene of Avian Myeloblastosis virus (AMV) which causes myeloblastic leukemia in chickens and transforms myelomonocytic cells in culture (Baluda et al., Virology 15: 185-199 (1964); C. Moscovici, Immunol. 71: 79-101 (1975)). The normal cellular counterpart of this oncogene, c-myb, is highly conserved and is present in all vertebrate and some invertebrate species examined (Franchini et al., Proc. Nat. Acad. Sci. USA 80: 7385-7389 (1983); Katzen et al., Cell 41: 449-456 (1985)). Proteins encoded by the viral as well as the cellular myb gene appear to be localized in the nucleus, and these proteins exhibit a sequence-specific DNA-binding activity (Klempnauer et al., Cell 37: 537-547 (1984); Boyle et al., Proc. Nat. Acad. Sci. USA 81: 42654269 (1984); Moelling et al., Cell 40: 983-990 (1985); Biedenkapp et al., Nature 335: 835-837 (1988)). Their sequence-specific DNA binding activity and ability to activate transcription of reporter genes linked to certain promoter/enhancer sequences suggest that they act as nuclear transcription factors (Sakura et al., Proc. Nat. Acad. Sci. USA 86: 5758-5762 (1989); Dudek et al., Proc. Nat. Acad. Sci. USA 89: 1291-1295 (1992)). A-myb in particular has been recognized as a potent transactivator of transcription (Golay et al., Oncogene 9: 2469-2479 (1994); Foos et al., Oncogene 9: 2481-2488 (1994)). Elimination of c-myb function in vivo, using gene-knock out techniques, has indicated that homozygous c-myb mutant mice fail to show effective fetal hepatic hematopoiesis resulting in the death of mice in utero confirming an essential role for c-myb in fetal hematopoiesis (Mucenski et al., Cell 65: 677-689 (1991)).
In contrast to c-myb, whose role in hematopoiesis is well established, little is known about the role of the A-myb gene in development. Human A-myb is expressed in a variety of lymphoid and solid tumors (Shen-Ong et al., Mol. Cell. Biol. 6: 380-392 (1986)). Foos et al., Oncogene 9: 2481-2488 (1994) have reported ubiquitous expression of A-myb in chicken cell lines. On the other hand, Sleeman, Oncogene 8: 1931-194 (1993) reported specific expression of Xenopus A-myb in testis, with very low levels of expression in ovarian tissue.
Murine spermatogenesis is divided into three distinct intervals which include: (1) stem cell proliferation and renewal; (2) meiosis and (3) germ cell differentiation (spermatogenesis). Spermatogenesis in mice occurs in the seminiferous tubule, a specialized epithelium in which spermatogonia are located in close proximity to the basement membrane. Cells at progressively later stages of meiosis and differentiation are situated closer to the tubular lumen. Spermatogenesis in the mouse occurs in twelve distinct histological stages. Each stage consists of a constant pattern of germ cell association. Stage VII of mouse spermatogenesis is a testosterone dependent stage and includes the following cell types: Type A spermatogonia (stem cells) along with preleptotene spermatocytes, usually situated closest to the basement membrane; pachytene spermatocytes (early meiotic cells) located at the intermediate position between basement membrane and the lumen; and step 7 spermatids and step 16 spermatozoa located closest to the lumen.
Recently, it has been demonstrated that A-myb is expressed at high levels in mouse testis where it is transcribed as multiple transcripts, some of which are differentially spliced to code for smaller proteins (Mettus et al., Oncogene 9: 3077-3086 (1994)). A high level of expression of A-myb was seen in mouse testis and very low levels of expression were detected in mouse spleen, ovary and brain. As differentiation proceeds and the primary spermatogonia mature into secondary spermatogonia, which in turn maturate into spermatocytes, a distinct downregulation of A-myb expression was seen in in Situ hybridization studies. A-myb was maximally expressed in type A spermatogonia which are located proximal to the basement membrane and preleptotene and pachytene spermatocytes located between the basement membrane and the lumen. Less intense hybridization was also seen with spermatids. Thus, A-myb expression was maximal in proliferating stem cells and early meiotic cells but reduced in spermatids and absent in spermatozoa undergoing terminal differentiation.
Despite these findings, the functional significance of A-myb remains to be established, particularly in spermatogenesis. More complete information concerning the function of A-myb requires studying the effect of the encoded protein, or the lack thereof, in vivo.
Various animals have been produced with germ line foreign DNA, or with altered levels of expression of certain genes. These animals typically have a foreign or mutated gene incorporated into their genome. In one such class of transgenic animal, the so-called homozygous null or xe2x80x9cknockoutxe2x80x9d mutants, expression of an endogenous gene has been suppressed through genetic manipulation.
Transgenic animals generally harbor at least one copy of a transgene either homologously or nonhomologously integrated into an endogenous chromosomal location so as to encode a foreign or mutant protein. Such transgenic animals are usually produced by introducing the transgene or targeting construct into a fertilized egg, or into an embryonic stem (ES) cell which is then injected into an embryo. Introduction of the transgene into the fertilized egg or ES cell is typically performed by microinjection, retroviral infection, electroporation, lipofection, or biolistics. The fertilized egg or embryo is then transferred to an appropriate pseudopregnant female for the duration of gestation. Knockout mutants may be obtained according to this method where the non-native DNA which is introduced comprises a nucleic acid construct that will be used to suppress expression of a particular gene. Such knockout constructs are typically introduced into ES cells.
One problem in the production of transgenic animals is the relatively low rate of success in obtaining incorporation of the transgene into the germline of the host species. Moreover, while transgenes have been incorporated into fertilized eggs by microinjection, the smaller size of sperm cells makes incorporation of transgenes by injection difficult. What is needed is a method to increase the frequency of first generation transgenic offspring and to provide for the incorporation of transgenes into sperm.
Male infertility continues to be significant reproductive health problem. What is needed is a live animal model which may be used for the study of male infertility, and for screening and evaluation of potential therapeutic agents useful in the treatment of this disorder.
Accordingly, it is an object of the invention to provide nonhuman animals in which expression of the A-myb gene has been suppressed.
It is an object of the invention to provide nonhuman cells and nonhuman animals containing a homozygous null mutation of the A-myb gene locus.
It is an object of the invention to provide constructs and vectors for producing such cells and animals containing an A-myb homozygous null mutation.
It is a further object of the invention to provide a method for obtaining incorporation of transgenes of interest into sperm cells, and to provide sperm cells so transformed.
It is an object of the invention to provide a method for the production of nonhuman transgenic animals, by utilizing the aforesaid transgenic sperm.
These and other objects of the invention will be apparent to those of ordinary skill in the art from the following disclosure.
In accordance with the foregoing objects, the invention in one aspect is a targeting construct for functionally disrupting an A-myb gene. The targeting construct comprises a polynucleotide containing at least one portion having a sequence that is substantially homologous to a sequence present in or flanking an A-myb gene locus and which, when integrated at the corresponding A-myb gene locus, functionally disrupts expression of A-myb protein from the gene locus. Such targeting constructs, or portions thereof, integrate at the A-myb gene locus by homologous recombination between the endogenous gene locus and the targeting construct.
In one embodiment, the A-myb gene is functionally disrupted by a targeting construct which inserts a sequence, typically into a coding sequence (i.e., exon), wherein the resultant disrupted A-myb gene is substantially incapable of expressing a functional A-myb protein. In one such embodiment, the targeting construct comprises an upstream homology region having a sequence with substantial identity to a first endogenous A-myb gene sequence, a nonhomologous replacement portion, a downstream homology region having a sequence with substantial identity to a second endogenous A-myb gene sequence located downstream from said first endogenous A-myb sequence, wherein the upstream homology region and downstream homology region flank the nonhomologous replacement portion.
The nonhomologous replacement portion of the targeting construct advantageously comprises a positive selection expression cassette, such as neo. The targeting construct further advantageously comprises a negative selection cassette distal to either the upstream homology region or the downstream homology region. The negative selection cassette may comprise, for example, a tk gene.
According to another embodiment, the invention provides a method for generating stem cells having a functionally disrupted endogenous A-myb gene comprising transferring the aforesaid targeting construct into pluripotent stem cells, and selecting for stem cells having a correctly targeted homologous recombination between the targeting construct and an endogenous A-myb gene sequence.
According to yet another embodiment, the invention provides a method for generating nonhuman animals having a functionally disrupted endogenous A-myb gene, comprising the steps of transferring, into a nonhuman blastocyst, stem cells having a correctly targeted homologous recombination between the aforesaid targeting construct and an endogenous A-myb gene sequence; implanting the resultant blastocyst into a pseudopregnant female; and collecting offspring harboring an endogenous A-myb allele having the correctly targeted homologous recombination.
According to-another embodiment, the invention provides transgenic nonhuman animals and stem cells having a genome comprising at least one functionally disrupted A-myb gene. The animal or stem cell is preferably homozygous for the functionally disrupted A-myb gene. Such a homozygous transgenic animal or stem cell is substantially incapable of directing the efficient expression of endogenous A-myb. For example, in a preferred embodiment, a transgenic mouse is homozygous for an inactivated endogenous (i.e., naturally occurring) A-myb gene.
According to one embodiment, the transgenic nonhuman animal or stem cell homozygous for a functionally disrupted A-myb gene comprise an A-myb gene disrupted by an integrated targeting construct, e.g., an integrated targeting construct comprising a neo gene.
According to a preferred embodiment of the invention, the transgenic animal is a mouse comprising a genome having a functionally disrupted murine A-myb allele. Preferably, the mouse is homozygous for the functionally disrupted A-myb allele. Such mice do not produce functional A-myb protein and are infertile.
According to another embodiment, a method is provided for generating nonhuman animals producing sperm harboring a desired transgene.
Spermatogonia are obtained from a nonhuman animal which is homozygous for a functionally disrupted endogenous A-myb gene. An A-myb construct comprising a first DNA sequence encoding a functional A-Myb polypeptide and a second DNA sequence encoding the desired transgene of interest, is transferred into the spermatogonia. The spermatogonia harboring the A-myb construct are then introduced into the testes of nonhuman animals which are homozygous for a functionally disrupted endogenous A-myb gene. Fertile individuals are then selected from the animals having received the transfected spermatogonia. Fertile individuals produce sperm harboring the desired transgene.
According to another method for generating nonhuman animals producing sperm harboring a desired transgene, the testis of nonhuman animals which are homozygous for a functionally disrupted endogenous A-myb gene are infected with an expression vector directing the incorporation into the DNA of said infected testes a first DNA sequence encoding a functional A-Myb polypeptide and a second DNA sequence encoding the desired transgene of interest, linked to said first DNA sequence. Fertile individuals are selected from the infected animals. The fertile individuals produce sperm harboring the desired transgene.
The present invention also provides for the treatment of male infertility in those occurrences of the disease which arise from a defect in the A-myb locus. Treatment comprises the transfer of DNA encoding functional A-Myb polypeptide to the cells of the testes, or by administration of functional A-Myb polypeptide directly to the testes.
According to one such treatment method for restoring fertility in a subject who is infertile due to a defect in the A-myb locus, spermatogonia is first obtained from the subject. An A-myb construct is transferred into the obtained spermatogonia. The A-myb construct comprises a DNA sequence encoding a functional A-Myb polypeptide. The spermatogonia harboring the A-myb construct encoding the functional A-Myb polypeptide is introduced into the testes of the individual to obtain production of functional A-Myb polypeptide in the testes.
In another embodiment of an infertility treatment method, fertility in a subject who is infertile due to a defect in the A-myb locus is restored by infecting the testis of the individual with a retrovirus vector. The vector directs the incorporation of a DNA sequence encoding a functional A-Myb polypeptide into the DNA of the infected testes.
In yet another embodiment, fertility is restored in a subject who is infertile due to a defect in the A-myb locus by locally administered a functional A-Myb polypeptide to the testes of the subject, such as by injection into the seminiferous tubules.
The invention is also directed to spermatogonia comprising recombinant DNA encoding a functional A-Myb polypeptide.
As used herein, the term xe2x80x9cA-myb genexe2x80x9d or xe2x80x9cA-myb gene locusxe2x80x9d refers to a region of a chromosome spanning all of the exons which potentially encode the A-myb polypeptide and extending through flanking sequences (e.g., including promoters, enhancers, etc.) that participate in A-myb protein expression. Thus, an A-myb gene locus includes the region spanning from the first exon through the last exon and also includes adjacent flanking sequences (e.g., polyadenylation signals) that may participate in A-myb gene expression.
The terms xe2x80x9cfunctional disruptionxe2x80x9d or xe2x80x9cfunctionally disruptedxe2x80x9d as used herein means that a gene locus comprises at least one mutation or structural alteration such that the functionally disrupted gene is substantially incapable of directing the efficient expression of functional gene product. By way of example but not limitation, an endogenous A-myb gene that has a neo gene cassette integrated into an exon of an A-myb gene is not capable of encoding a functional A-myb protein and is therefore a functionally disrupted A-myb gene locus. Deletion or interruption of essential transcriptional regulatory elements, polyadenylation signals(s), splicing site sequences will also yield a functionally disrupted gene. Functional disruption of an endogenous A-myb gene, may also be produced by other methods (e.g., antisense polynucleotide gene suppression). The term xe2x80x9cstructurally disruptedxe2x80x9d refers to a targeted gene wherein at least one structural (i.e., exon) sequence has been altered by homologous gene targeting (e.g., by insertion, deletion, point mutation(s), and/or rearrangement). Typically, alleles that are structurally disrupted are consequently functionally disrupted. However A-myb alleles may also be functionally disrupted without concomitantly being structurally disrupted, i.e., by targeted alteration of a non-exon sequence such as ablation of a promoter. An allele comprising a targeted alteration that interferes with the efficient expression of a functional gene product from the allele is referred to as a xe2x80x9cnull allelexe2x80x9d.
The expression xe2x80x9cfunctional A-Myb polypeptidexe2x80x9d means a polypeptide which, upon expression in or administration to A-mybxe2x88x92/xe2x88x92 male individuals, is sufficient to restore spermatogenesis and fertility in such individuals.
The term xe2x80x9ccorresponds toxe2x80x9d is used herein to mean that a polynucleotide sequence is homologous (i.e., is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence.
The term xe2x80x9ccomplementary toxe2x80x9d is used herein to mean that the subject sequence is homologous to all or a portion of a reference polynucleotide sequence. For illustration, the nucleotide sequence xe2x80x9cTATACxe2x80x9d corresponds to a reference sequence xe2x80x9cTATACxe2x80x9d and is complementary to a reference sequence xe2x80x9cGTATAxe2x80x9d.
The terms xe2x80x9csubstantially corresponds toxe2x80x9d, xe2x80x9csubstantially homologousxe2x80x9d, or xe2x80x9csubstantial identityxe2x80x9d as used herein denotes a characteristic of a nucleic acid sequence, wherein a nucleic acid sequence has at least about 70 percent sequence identity as compared to a reference sequence, typically at least about 85 percent sequence identity, and preferably at least about 95 percent sequence identity as compared to a reference sequence. The percentage of sequence identity is calculated excluding small deletions or additions which total less than 25 percent of the reference sequence. The reference sequence may be a subset of a larger sequence, such as a portion of a gene or flanking sequence, or a repetitive portion of a chromosome. However, the reference sequence is at least 18 nucleotides long, typically at least about 30 nucleotides long, and preferably at least about 50 to 100 nucleotides long.
xe2x80x9cSubstantially complementaryxe2x80x9d as used herein refers to a sequence that is complementary to a sequence that substantially corresponds to a reference sequence. In general, targeting efficiency increases with the length of the targeting transgene portion (i.e., homology region) that is substantially complementary to a reference sequence present in the target DNA (i.e., crossover target sequence). In general, targeting efficiency is optimized with the use of isogeneic DNA homology clamps, although it is recognized that the presence of various recombinases may reduce the degree of sequence identity required for efficient recombination.
The term xe2x80x9cnonhomologous sequencexe2x80x9d, as used herein, has both a general and a specific meaning, it refers generally to a sequence that is not substantially identical to a specified reference sequence, and where no particular reference sequence is explicitly identified, it refers specifically to a sequence that is not substantially identical to a sequence of at least about 50 contiguous bases at an endogenous A-myb gene.
The term xe2x80x9cnaturally-occurringxe2x80x9d as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring. As used herein, laboratory strains of rodents which may have been selectively bred according to classical genetics are considered naturally-occurring animals.
As used herein, the term xe2x80x9ctargeting constructxe2x80x9d refers to a polynucleotide which comprises: (1) at least one homology region having a sequence that is substantially identical to or substantially complementary to a sequence present in a host cell A-myb gene locus, and (2) a targeting region which becomes integrated into an host cell A-myb gene locus by homologous recombination between a targeting construct homology region and said A-myb gene locus sequence. If the targeting construct is a xe2x80x9chit-and-runxe2x80x9d or xe2x80x9cin-and-outxe2x80x9d type construct (Valancius and Smithies (1991) Mol. Cell. Biol. 11: 1402; Donehower et al. (1992) Nature 356: 215; (1991) J. NIH Res. 3: 59; which are incorporated herein by reference), the targeting region is only transiently incorporated into the endogenous A-myb gene locus and is eliminated from the host genome by selection. A targeting region may comprise a sequence that is substantially homologous to the endogenous A-myb gene sequence and/or may comprise a nonhomologous sequence, such as a selectable marker (i.e., neo, tk, gkt). The term xe2x80x9ctargeting constructxe2x80x9d does not necessarily indicate that the polynucleotide comprises a gene which becomes integrated into the host genome, nor does it necessarily indicate that the polynucleotide comprises a complete structural gene sequence. As used in the art, the term xe2x80x9ctargeting constructxe2x80x9d is synonymous with the term xe2x80x9ctargeting transgenexe2x80x9d.
The terms xe2x80x9chomology regionxe2x80x9d and xe2x80x9chomology clampxe2x80x9d as used herein refer to a segment (i.e., a portion) of a targeting construct having a sequence that substantially corresponds to, or is substantially complementary to, a predetermined A-myb gene sequence, which can include sequences flanking said A-myb. A homology region is generally at least about 100 nucleotides long, preferably at least about 250 to 500 nucleotides long, typically at least about 1000 nucleotides long or longer.
The terms xe2x80x9ccrossover target sequencesxe2x80x9d or xe2x80x9cendogenous target sequencesxe2x80x9d as used herein refer to A-myb gene sequences that substantially correspond to, or are substantially complementary to, a transgene homology region.
As used herein, the term xe2x80x9ctargeting regionxe2x80x9d refers to a portion of a targeting construct which becomes integrated into an endogenous chromosomal location following homologous recombination between a homology clamp and an endogenous A-myb gene sequence. Typically, a targeting region is flanked on each side by a homology clamp, such that a double-crossover recombination between each of the homology clamps and their corresponding endogenous A-myb gene sequences results in replacement of the portion of the endogenous A-myb gene locus by the targeting region; in such double-crossover gene replacement targeting constructs the targeting region can be referred to as a xe2x80x9creplacement regionxe2x80x9d. However, some targeting constructs may employ only a single homology clamp (e.g., some xe2x80x9chit-and-runxe2x80x9d-type vectors, see, Bradley et al. (1992) Bio/Technology 10: 534, incorporated herein by reference).
As used herein, the term xe2x80x9creplacement regionxe2x80x9d refers to a portion of a targeting construct flanked by homology regions. Upon double-crossover homologous recombination between flanking homology regions and their corresponding endogenous A-myb gene crossover target sequences, the replacement region is integrated into the host cell chromosome between the endogenous crossover target sequences. Replacement regions can be homologous (e.g., have a sequence similar to the endogenous A-myb gene sequence but having a point mutation or missense mutation), nonhomologous (e.g., a neo gene expression cassette), or a combination of homologous and nonhomologous regions.